Corneal endothelial cells (CEnC) are critical for maintaining corneal transparency. Many factors, including aging, oxidative stress, and inflammation have been implicated in CEnC damage. CEnC loss also complicates eye banking where prolonged storage can lead to progressive decline in CEnC density, which can lead to graft edema. CEnC damage is also the chief proximate cause of graft failure, whether immune-driven or not. Thus, cytoprotective strategies that enhance CEnC viability could have a major impact on (i) the quality and number of donor corneas available for transplantation, and (ii) graft outcomes. Clinical data have suggested a correlation between nerve density and CEnC numbers; numerous conditions, including diabetes, dry eye, and herpetic keratitis, which induce nerve damage, are also associated with measurable CEnC loss. Procurement of donor corneas for eye banking also requires severing donor tissue from nerves, and is associated with significant CEnC loss. Our preliminary data show (1) high constitutive expression of melanocortin receptor (MCR) for the neuropeptide alpha-melanocyte stimulating hormone (?-MSH) in both human and murine CEnC; (2) significant suppression of CEnC death induced by inflammatory cytokines or the oxidant hydrogen peroxide by ?-MSH; and 3) decrease in eye banked CEnC loss when donor tissues are kept in medium supplemented with ?-MSH. Based on these preliminary data, we hypothesize that ?-MSH provides cytoprotection to CEnC from both oxidative stress and inflammatory cytokines, and thus predict that ?-MSH promotes survival of CEnC (i) in cornea storage and (ii) after transplantation. To test this hypothesis, we will pursue two specific aims. In Aim 1, we evaluate the effect of ?-MSH on CEnC loss in eye bank-stored corneas in collaboration with the Eversight Eye Bank. We will keep human donor corneas in either standard Optisol-GS medium (Aim 1A) or in medium supplemented with hydrogen peroxide to induce oxidant stress (Aim 1B) before adding ?-MSH or control PBS. Tissues will be monitored and assessed prospectively using standard eye bank methodologies, and tests for CEnC viability (TUNEL assay and cytochrome c release) and oxidative DNA damage. In Aim 2, we will assess the effect of ?-MSH on the function and survival of grafted CEnC in murine corneal grafting. To discriminate the potential immunomodulatory effect of ?-MSH from its cytoprotective effect, ?-MSH (or control treatment) will be used in syngeneic grafts in Aim 2A. Then, we will use a variety of MCR knockout combinations in hosts (Aim 2B) or donors (Aim 2C) to discriminate the cellular targets of ?-MSH. This grant, which represents a new area of investigation for our lab, brings together synergy between our lab with core expertise in transplantation, with an investigative group that includes experts on CEnC (Dr. Jurkunas) and neuropeptide biology (Dr. Taylor). We aim to employ data generated in this exploratory grant to generate a more mechanistically focused R01 application whose results can then be employed by eye banks for optimized tissue preservation.